Duocarmycin SA (1)1 and CC-1065 (2)2 are two parent members of a class of highly potent naturally occurring antitumor agents that also include duocarmycin A3 and yatakemycin4 (FIG. 1). This unique class of natural products derives its antitumor properties from their ability to alkylate DNA in a sequence selective manner.5,6 Comprehensive studies of the natural products, their synthetic unnatural enantiomers,7 and key analogues have defined many of the fundamental features that control the DNA alkylation selectivity, efficiency, and catalysis, resulting in a detailed understanding of the relationships between structure, reactivity, and biological activity.6,7,8 
CBI (1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one):
is one of the most extensively studied synthetic analogues of the family since we first introduced it in 1989.9 The CBI alkylation subunit is not only more synthetically accessible and participates in the now characteristic DNA alkylation reaction effectively,10 but it has also been found to be 4 times more stable and 4 times more potent than the naturally occurring alkylation subunit of CC-1065 (2), approaching the stability and potency of the duocarmycin SA (1) alkylation subunit. Since analogues incorporating the CBI alkylation subunit have also been established to exhibit efficacious in vivo antitumor activity in animal models, it is an excellent synthetic replacement on which to examine the structure-function features of the natural products, including new prodrug design and evaluation.11

During the course of the total syntheses of CC-1065 (2), duocarmycin SA (1), duocarmycin A, yatakemycin, and related analogues including CBI-indole2 (5),11c it was established that the synthetic phenol precursors such as 4, which have yet to undergo the Winstein Ar-3′ spirocyclization, are equipotent to and indistinguishable from their cyclized cyclopropane containing counterparts within in vitro cytotoxic assays, DNA alkylation studies, and in vivo antitumor models.

Due to this indistinguishable behavior both in vitro and in vivo and because their extraordinary potency creates special precautions for their handling, protection of the phenol precursors not only permits safe handling during their preparation, but it also provides an effective site on which to create prodrugs that can be designed for controlled release in vivo.12 Such prodrugs incorporating phenol acylation have been developed to simultaneously improve solubility, pharmacokinetics, storage life, handling safety, and efficacy in vivo.12,13,14 Two such carbamate-based drugs, KW-218912c-d(t1/2=20 h, calf serum) and carzelesin (U-80,244, t1/2<1 h, human plasma),12a-b which are rapidly cleaved in vivo (1-20 h), entered clinical trials but have ultimately not progressed. In related studies, we described ester and carbamate prodrugs 3a-f of (+)-CBI-indole2, many of which were found to be essentially equipotent to (+)-CBI-indole2 (5) in vitro.12e However, upon hydrolysis, such prodrug compound necessarily release a byproduct (shown as RN—H below) as well as the active drug in vivo, which can be a cause of concern with respect to possible byproduct toxicity.
This work established that the free drug is rapidly released in a cellular assay and is able to spirocyclize, alkylate DNA, and express its biological activity efficiently in a manner essentially indistinguishable from the free drug itself.